Jumat, 14 Juni 2013

Aspergillus Oryzae



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Aspergillus oryzae is one of the ascomycetous fungus that asexual species. Its used in the production of soy sauce, miso and sake without recorded incidents  for hundreds of years. There are conflicting opinions about whether A. oryzae can be isolated in nature. Although the details of the genetic relationship between A. oryzae and A. flavus remain unclear, the two species are so closely related that all strains of A. oryzae are regarded by some as natural variants of A. flavus modified through years of selection for fermenting of foods. A. oryzae is regarded as not being pathogenic for plants or animals, though there are a handful of reports of isolation of A. oryzae from patients. There are also several reports of products of A. oryzae fermentations, e.g. a-amylase, that seem to be associated with allergic responses in certain occupations with high exposure to those materials. A. oryzae can produce a variety of mycotoxins when fermentation is extended beyond the usual time needed for production of these foods. While wild A. flavus isolates readily produce aflatoxins and other mycotoxins, A. oryzae has not been shown to be capable of aflatoxin production.
Aspergillus oryzae has apparently been an essential part of oriental food production for centuries and is now used in the production of many different oriental foods such as soy sauce, sake and miso. Potential uses under TSCA include fermentations of numerous enzymes, e.g., amylase, protease, B-galactosidase, lipase, and cellulase, and organic compounds such as glutamic acid. While these products have a variety of potential commercial uses, some of them are mostly frequently used in food processing.
The experience of safe commercial use of A. oryzae is extraordinarily well established. As a "koji" mold it has been used safely in the food industry for several hundred years. A. oryzae is also used to produce livestock probiotic feed supplements. Even the commercialization of byproducts of the fermentation was established nearly a century ago. The "koji" mold enzymes were among the first to be isolated and commercialized. In 1894, Dr. J. Takamine isolated and soldTakadiastase from a commercial firm he started in Clifton, New Jersey (Bennett, 1985a).
EPA has reviewed, under TSCA, two genetically modified strains of A. oryzae used for the production of enzymes (Premanufacture Notice (PMN) numbers P89-134 and P94-1475). The candidate species is a member of the genus Aspergillus and belongs to the group of fungi that are generally considered to reproduce asexually (Fungi Imperfecti or Deuteromycetes), although perfect forms (forms that reproduce sexually) of some aspergilli have been found. The form genus Aspergillus represents a taxonomic grouping of a very large number of asexual fungi which are characterized by the production of spores on large black or brown conidia in phialides arranged on a characteristic spherical conidiophore termed the vesicle. This definition leads to inclusion of a complex assortment of organisms within the taxon. To simplify the taxonomy of such a large number of organisms, the genus Aspergillus has been divided into sections or groups based on color, size and roughness of the spore, conidiophore and vesicle as well as the arrangement of phialides and the presence of sclerotia. The separation of individual species into groups is somewhat tenuous and based on distinguishing measured characters with overlapping means. This resulted in the 132 species arranged in 18 groups by Raper and Fennell (1965) due to overlapping morphological or physiological characteristics. However, it is important to remember that taxonomy is "dealing with living variable organisms and that species and group concepts must be reasonably elastic" (Raper & Fennell, 1965).
As is the case of many fungi, the taxonomy of Aspergillus is primarily based on morphological features, rather than physiological, biochemical features and genetic characteristics often used to classify bacteria. Nomenclature problems of the genus Aspergillus arise from their pleomorphic life cycle. The newer findings show that this group of fungi has both a perfect (teleomorphic) and an imperfect (anamorphic) state.
The morphological approach to taxonomy has led to the existence of several synonyms for the genus Aspergillus. They are: Alliospora Pim; Aspergillonsis Spegazzini; Cladaspergillus Ritg; Cladosparum Yuill and Yuill; Euaspergilus Ludwig; Gutturomyces Rivolta; Raperia Subramaniam and Grove; Sceptromyces Corda; Spermatoloncha Spegazzini; Sphaeromyces Montagne; Sterigmatocystis Cramer; and Stilbothamnium Hennings (Bennett, 1985b). Aspergilli are ubiquitous in nature. They are geographically widely distributed and have been observed in a broad range of habitats, because they can colonize a wide variety of substrates.
Aspergillus oryzae is a member of the A. flavus group of Aspergillus species. The A. flavus group, which also now includes A. sojae, A. nomius and A. parasiticus (see below) is defined by the production of spore chains in radiating heads which range in color from yellow-green to olive brown. The conidiophores are roughened and colorless. The spores themselves have conspicuous ridges and echinulations (spines). Sclerotia are occasionally produced (Raper & Fennell, 1965). A. oryzae/flavus species have never been connected to a sexual or teleomorphic stage. However, the teleomorphic stages of other Aspergillus species have been demonstrated by the formation of cleistothecia. These species belong to the genera Emericella, Neosartorya and Eurotium, all belonging to the ascomycetous family Eurotiaceae (Fennel, 1973). Either the sexual stages of the A. flavus group have not been recognized as such, being identified as completely different species based on morphology, or this group of fungi are "degenerate", having lost the ability to form sexual spores and mycelia.
A. oryzae is considered by some experts to be a domesticated variant of A. flavus (Kurtzman et al. 1986). Through long-time use, A. oryzae strains seem to have been selected to exhibit reduced sporulation, have more aerial mycelia and exhibit no environmental survival structures like sclerotia or the presence of aflatoxins that might function to inhibit grazing by insects. These morphological features that differentiate A. oryzae from A. flavus may represent adaptations to the artificial culture conditions of the koji fermentation. Misidentification of new isolates not obtained from well established cultures is always a possibility, since the key morphological differences between the two species seem related to culture adaptation. However, the source of A. oryzae strains for industrial fermentations today is likely to be standard culture collections. Environmental isolates of aspergilli would likely be identified as A. flavus rather than the laboratory-adapted A. oryzae.
The taxonomy of Aspergillus has public health implications due to the production of potent mycotoxins by members of this genus. Most notable is the association of aflatoxins with members of the A. flavus group (Bennett, 1985b; Semeniuk et al., 1971). A. oryzae is a member of that group and in spite of the above mentioned morphological distinctions, A. oryzae appears to be very closely related to A. flavus. Numerous studies have beendone to distinguish the koji molds from their toxicogenic relatives. The results are unambiguous in their confirmation of the conspecificity of A. oryzae and A. flavus. (see Section IV. below).
In a similar way, A. sojae is considered to be a domesticated form of A. parasiticus and shares a 92% DNA homology with its wild progenitor. A. sojae also has a history of safe use in the food industry. A. parasiticus in nature is an active colonizer of cereal grains and seeds with concurrent mycotoxin production. While these species can be distinguished from A. flavus/oryzae using morphological criteria, all four species intergrade. The hazard concerns for these species, thus, are equivalent to those associated with A. flavus/oryzae.
A. nomius is a newly classified species of toxigenic strains originally described in the A. flavus group, but not having the same level of DNA homology as shown among the four varieties mentioned above (Kurtzman et al., 1987). A. nomius produces aflatoxin and includes strains isolated from diseased bees. A. oryzae is distinguishable both morphologically and genetically from A. nomius. The close relationship between A. oryzae and A. flavus and the production of highly toxic mycotoxins by the latter has resulted in careful examination of the toxigenic potential of A. oryzae. However, A. oryzae, as a koji mold, has toxigenic potential in its own right. Those aspergilli used for manufacture of Japanese fermented foods have long been called koji molds. Prominent among the 25 koji molds listed is A. oryzae (Manabe et al., 1984). This fungus is used for sake, an alcoholic beverage, miso a soy bean paste, shoyu, soy sauce, amasake, a sweet beverage, and shouchu, a distilled liquor.
A. flavus commonly colonizes damaged cereal grains, soybeans and peanuts, actively producing mycotoxins (Stoloff, 1982). Certain strains of A. oryzae have themselves been shown to produce the mycotoxins aspergillic, kojic, cyclopiazonic and B-nitropropionic acids and maltoryzine (Ciegler & Vesonder, 1987). Even with the food industry strains, a caveat of safety is that the fungal incubation not exceed the normal three day period. A. oryzae has been shown to produce toxic compounds under incubations longer than the typical koji fermentation(Semeniuk et al, 1971; Yokotsuka & Sasaki, 1986). The following are toxins produced by some strains of A. oryzae.
Kojic acid (discovered by Saito, 1907) is produced by koji, a solid culture of the koji mold. It is a commonly produced metabolite that possesses antibacterial and antifungal activity. Few oral studies exist for this byproduct. Giroir reported toxic effects on chickens at four to eight mg/kg feed. Older studies (Friedemann, 1934, Werch et al. 1957, Morton et al., 1945) using intravenous or intraperitoneal challenges show moderate toxicity for kojic acid. Later work had similar results (Ueno and Ueno, 1978). Kojic acid also is reported to have moderate cardiotoxic and cardiotonic activity (Manabe et al., 1984., Bajpai et al. 1982). Nineteen of 47 A. oryzae strains tested produced kojic acid (Manabe et al., 1984). Even though it is apparent that the koji molds, including A. oryzae can produce the toxin kojic acid, this toxin may not be present in the fermented foods. The incubation period for sake, shoyu and miso is about two days and no kojic acid is found at that time (Manabe et al., 1984). However, these authors concluded that they were unable to prove kojic acid was not present in any fermented food in Japan, because conditions of production and materials were different for each industry, and were often uncontrolled. Semeniuk et al. (1971) warned that even with food industry strains, fungal incubation must not exceed three days. Thus, as the culture adjusts to changing conditions, A. oryzae may produce toxic compounds when incubation time exceeds typical koji fermentation time.
Maltoryzine, another toxic metabolite isolated and characterized by Iisuka and Iida (1962), was produced by A. oryzae var. microsporus. This metabolite was determined to be the cause of poisoning among dairy cows. While highly toxic (LD50 3 mg/kg; Iizuka, 1974; Ciegler and Vesonder, 1987), the substance may only be found in one or a very few strains of A. oryzae. The single isolate, IAM 2950, produced enough of the toxin when grown on malt rootlets to poison some milk cows, prompting the determination of its LD50. The production of these toxins is related to the composition of the growth substrate and usually occurs in stationary phase cultures. Commercial strains of A. oryzae and A. sojae apparently do not produce maltoryzine.
Pitt and Cruickshank (1990), note that many isolates of Aspergillus oryzae are found to produce cyclopiazonic acid. Orth (1977), reporting on food industry strains of A. oryzae, indicated that eight of 16 strains produced cyclopiazonic acid. This acid is a natural contaminant of foods and feeds and is produced by several molds including those used in fermented food production. These included A. flavus, A. versicolor, A. tamarii, several Penicillium species, including P. camemberti, and A. oryzae. This mycotoxin has been shown to occur naturally in corn, cheese, peanuts and in Kodo millet that was implicated in natural human intoxication in India (CAST Task Force Report No. 116, 1989a). Benkhemmar et al. (1985) showed that when cyclopiazonic acid producing (CPA+) strains are mated with CPA-strains, the CPA+ phenotype is dominant in the heterokaryon. Oral administration produced effects at levels ranging from 0.25 to >50 mg/kg with dogs among the most sensitive species and rats among the least (Purchase, 1971; Nuehring et al., 1985). LO(A)ELs for sensitive species were at or under 1mg/kg. Nishie et al. (1985) noted that Rao and Husain (1985) identified cyclopiazonic acid as the cause of debilitating illnesses in cattle and man in India.
A. oryzae can produce b-nitropropionic acid, along with other food-borne molds (Gilbert et al., 1977). Its mode of action is apparently irreversible succinate dehydrogenase inhibition which can cause a variety of symptoms often neurological in nature. These symptoms have been studied in mice (Gould and Gustine, 1982; Umezawa, 1967) and rats (Hamilton and Gould, 1987) where intravenous or subcutaneous LD50s of 20-50 mg/kg were determined. Reports of livestock poisoning via ingestion in feed (James et al., 1980; James, 1983) showed that ingestion of b-nitropropionic acid could produce significant toxic effects up to and including death. When A. oryzae (ATCC 12892) was studied for its ability to produce b-nitropropionic acid on various high protein and carbohydrate-rich foods, it flourished and produced this toxin in cooked sweet potato, potato and ripe banana (Penel and Kosikowski, 1990). Ames type assays for mutagenicity (Dunkel, 1985) showed positive responses with and without activation for two Salmonella strains, but not for three others. This assay uses multiple indicator strains in order to ensure that each potential mutation mode is detectable; the failure in three strains merely implies that the mutation modes to which each is sensitive are not the ones associated with the test substance.
The closest taxon to A. oryzae is A. flavus which Kurtzman et al. (1986) regard as conspecific. Many strains of A. flavus produce aflatoxins which are acutely toxic to mammals (oral LD50s ranging from 1 to 15 mg/kg depending on test species (Ceigler, 1975). Aflatoxins are animal carcinogens (Barnes and Butler, 1964; Dickens and Jones, 1964; Sinhuber, 1968) and also probablehuman carcinogens (Council for Agricultural Science and Technology, 1989). Developmental effects have also been found (Elis and DiPaolo, 1967, Le Breton et al., 1964).
While the koji molds like A. oryzae are distinguishable from, they are nevertheless very closely related to, A. flavus. Distinguishing between A. oryzae and A. flavus by physical traits is elusive. The toxigenic subspecies/variety A. flavus has numerous spores chains that remain yellow-green; sterigmata that are always biseriate; spiny (echinulate) individual spores; roughened conidiophores up to 600æm in length and sclerotia often present. The variety called A. oryzae specifically has fewer spore chains, fading to brown with age; longer average conidiophores (about two to three mm); smoother individual spores; sterigmata usually in 1 series and sclerotia rarely produced (Raper & Fennel, 1965).
Despite this strong similarity between the two species, production of aflatoxins has not been demonstrated by A. oryzae. Many studies affirm that the currently available strains confirmed to be A. oryzae are not capable of producing aflatoxins (Wei and Jong, 1986; Yokotsuka and Sasaki, 1986). In one test, no strains of A. oryzae or A. sojae (another koji mold) produced detectable levels of aflatoxins, while 33% and 85% of the strains of A. flavus and A. parasiticus, respectively, were toxigenic. As mentioned above, Kurtzman, et al. (1986) regard A. oryzae and A. sojae as domesticated varieties of their respective subspecies. Only one study (El-Hag and Morse, 1976) describes aflatoxin production by a strain reported to be Aspergillus oryzae (NRRL strain 1988). This observation is notable as an exception to the rule of no aflatoxin production by A. oryzae.
It has been noted that A. flavus strains upon extended laboratory cultivation lose morphologically distinguishing characteristics, making them appear much like A. oryzae (Kurtzman, et al., 1986). Wicklow (1984) details the competitive disadvantages of A. oryzae and implies that A. flavus is the "wild" form. Kurtzman et al. (1986) ask whether the separation between toxigenic and non-toxigenic A. flavus group species occurs through ecological adaptation or chromosomal changes such as translocations or inversions.

The elucidation of metabolic pathways responsible for the production of aflatoxins by A. flavus group fungi has progressed rapidly. Recently Payne (Bhatnagar, et al. 1992 and Payne, 1994) reported on the conversion of an aflatoxin non-producing strain of A. flavus to aflatoxin B1 positive using a cosmid library developed from a toxigenic A. flavus. While added metabolic precursors could not stimulate toxin production in the mutant, the addition of an appropriate cosmid carrying a <5 kb fragmentof the genome of the toxin producer converted the non-toxigenic strain to significant levels of aflatoxin production. Further work has resulted in isolation of a small segment specifying a regulatory, rather than structural, gene that affects early parts of the pathway. Probes for this regulatory gene, designated afl R, have been positive in both A. oryzae and A. sojae, even though those strains do not produce aflatoxin. In addition, Payne stated that probes for structural genes for aflatoxin production were also positive in some, but not all, A. oryzae strains examined.
It appears that evidence is mounting towards multiple reasons for failure to produce aflatoxins in A. oryzae cultures. One explanation is a lack of functional regulators, specifically afl R, that activate aflatoxin production. Another is that some or all of the structural genes in the aflatoxin pathway may be non-functional. For both types of genes, those sequences could be absent or present in the wrong orientation or split by insertions or modified slightly so as to be non-functional. Except for substantial deletion or absence of the necessary sequences, all of these alternatives are potentially reversible. However, Payne indicated that he doubted that industrial strains of A. oryzae were likely to revert to aflatoxin production. He indicated that, even though probes found the presence of appropriate gene sequences, the genes so detected could easily be incomplete enough so as to be completely non-functional.
Thus, complete absence of genetic potential is not the only plausible explanation for the non-expression of characters such as aflatoxin production in A. oryzae. In a related study, researchers attempting to improve strains of a mold identified as A. oryzae used for food fermentation in Thailand acquired a toxin producing strain by simple UV mutagenesis of a known "safe" strain (Kalayanamitr, et al. 1987). The toxins produced by this strain and other toxigenic A. oryzae strains are not aflatoxins but rather other types of mycotoxins. The exact composition of the toxins involved in A. oryzae toxicosis in these studies, as in other anecdotal studies, was not determined (Semeniuk, et al., 1971; Wicklow and Dowd, 1989, and Kalayanamitr, et al., 1987). The mechanism for this conversion to toxigenicity was not investigated, but the mutations required could have affected either structural or regulatory genes and produced the new observed toxigenic phenotype.
Aspergillus oryzae does not appear to be a human pathogen. Available information documents infections in humans possibly caused by A. oryzae in only three instances. The first was a case of meningitis (Gordon, et al., 1976). In the second case, A. oryzae invaded the paranasal sinuses, causing fever and right periorbital swelling (Byard, et al., 1986). The third case was apulmonary aspergilloma caused by A. oryzae (Liao, 1988). Care must be exercised in evaluating these three cases as having been caused by this organism due to its close taxonomical relationship to A. flavus and the possibility of incorrect identification. The relative rarity of such cases in light of the commonplace use of A. oryzae suggests this species has a low potential for expressing pathogenic traits.
Allergic reactions are not uncommon for aspergilli in general. There is one reported case of an allergic bronchopulmonary aspergillosis due to A. oryzae in a 19-year old female (Akiyama et al., 1987). However, the a-amylase produced by A. oryzae, that is used by bakers in bread making, was reported by Birnbaum, et al. (1988) to have caused asthma in a baker. Based on an observation of a case of baker's asthma due to monovalent sensitization to a-amylase used as an additive to flour, investigators tested 31 bakers who had occupational asthma and/or rhinitis by skin tests and serologic RAST examinations. Thirty-two percent of the bakers had RAST specific IgE to a-amylase from A. oryzae. Baker's asthma is reported to be the most frequent occupational lung disease in Switzerland and West Germany (Wuthrich and Baur, 1990). However, allergic reactions in bread bakers are quite common, both to the flours of various grains, as well as to the flour additives such as fungal amylases. Allergic reactions in bakers are not specific to A. oryzae, nor the enzymes produced by A. oryzae (O'Neil and Lehrer, 1991). In addition, the exposure scenario of a bread baker to flour and the additives contained therein is quite different from that of workers in a fermentation facility using general worker hygiene and protection practices.
There are two possible concerns for human health hazards associated with A. oryzae. The first, which is directly tied to A. oryzae, is the potential for mycotoxin production with extended fermentation. A variety of toxins can be produced, with the most common being the moderately toxic kojic acid. Other more potent toxins may only be produced by a few strains or in lesser quantities. These mycotoxins seem to be produced only under conditions of extended fermentation, and therefore, their production could be averted under proper fermentation conditions i.e., short fermentation times.

The second issue is the possibility for the production of aflatoxins because of the nearly indistinguishable identity of A. oryzae and A. flavus. Kurtzman, et al. (1986) have shown that A. flavus and A. oryzae are essentially the same based on DNA comparisons. A. oryzae appears so closely related to its aflatoxin-producing counterpart as to be viewed as consisting ofculture-attenuated strains of A. flavus (Kurtzman, 1994; Wicklow, 1984). It has been hypothesized that A. oryzae evolves under culture from A. flavus strains due to selection for features that would be ecologically detrimental in the wild.
Hypothetically, then, if A. oryzae has evolved to non-aflatoxigenic status after centuries in culture, the question remains whether it can revert to the "wild" type. The experience of oriental food production would seem to suggest not, or at least not frequently enough as to be detectable. Recent studies (Payne, 1994; Klich, 1994) suggest homology between parts of the A. oryzae genome and structural genes for aflatoxin production. It is conceivable that reintroduction of regulatory genes or their gene products could activate a dormant aflatoxin synthetic potential. There is no evidence to show that the required gene transfer or gene rearrangement that might provide the needed functional sequences for an aflatoxin producing A. oryzae strain occurs naturally. The question is, therefore, whether this type of genetic modification is possible in culture. Gene transfer from a toxigenic strain during fermentation is highly unlikely due to the need for maintaining axenic conditions during fermentation. The theoretical possibility of genetic rearrangement occurring in culture resulting in reversion back to the "wild-type" seems unlikely. Anecdotal evidence gathered over centuries suggests that A. oryzae commercial food strains do not produce aflatoxins, nor have there been reports of any adverse human health effects from aflatoxin.
The potential for toxin production is the main environmental hazard issue of concern for A. oryzae. If there were a method to distinguish between toxicogenic and non-toxicogenic strains, there would be no environmental concern for A. oryzae. Two recent studies that addressed the question of differentiating between toxin producing and non-toxicogenic strains of the related species A. flavus, A. parasiticus and A. nomius were unable to correlate either mitochondrial or chromosomal DNA RFLPs with mycotoxin production (Moody & Tyler, 1990a, 1990b). This again points to differences that may only involve small regulatory regions or that involve differences in structural gene complements that are beyond the detection limit of current DNA typing technologies.

Compounding this is the observation that A. oryzae and A. flavus are essentially indistinguishable by most molecular techniques. A. flavus is believed to be second in frequency only to the frank fungal pathogen, A. fumigatus, as a cause of aspergillosis in many species. A. flavus is associated specifically with invasive diseases of insects as well as toxicosis (Austwick, 1965). Recently, some insect pathogenic A.flavus strains were reclassified into A. nomius (Kurtzman et al., 1987). Whether A. oryzae is involved depends on how one defines the species of the A. flavus group. The effects on livestock of the various toxins that occur after extended koji fermentations, or in contaminated feed, show that the "minor" mycotoxins can still cause economic loss. No anecdotal accounts have been found that demonstrate that these potential effects occur in wildlife outside the agricultural environment. No reports of A. oryzae effects on living plants have been found. This species does not appear to be pose a hazard to plants.
The issues for environmental hazards are similar to those for human health hazards. The primary hazard concerns are for toxin production by A. oryzae strains. Under usual conditions of culture, well established commercial strains of this species do not seem to produce significant levels of mycotoxins, although certain moderately potent toxins can be produced after extended culture. Aflatoxins appear not to be produced by such cultures. The potential for environmental hazard is dependent on the likelihood that commercial strains could escape and establish themselves in the wild and grow under conditions analogous to those resulting in toxin production in extended culture. The few examples of livestock poisoning associated with the "minor" toxins, b-nitropropionic acid, maltoryzine and cyclopiazonic acid cited above imply that, for a short time at least, strains of A. oryzae may be able to survive in the wild.
Aspergillus oryzae is considered a Class 1 Containment Agent under the National Institute of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules (U.S. Department of Health and Human Services, 1986). In Europe, Aspergillus spp. are treated as low-risk-class microorganisms, i.e., category 2 of the European Federation of Biotechnology (Frommer et al., 1989) or category 1 on the OECD containment scale. Category 1 of the European Federation of Biotechnology scale includes organisms deemed harmless, which can be grown under good industrial large scale practices (GILSP), while category 2 organisms like Aspergillus require more stringent containment.

No data were available for assessing the release and survival specifically for fermentation facilities using A.oryzae. Therefore, the potential worker exposures and routine releases to the environment from large-scale, conventional fermentation processes were estimated on information available from eight premanufacture notices submitted to EPA under TSCA Section 5 and from published information collected from non-engineered microorganisms (Reilly, 1991). These values are based on reasonable worst-case scenarios and typical ranges or values are given for comparison.
During fermentation processes, worker exposure is possible during laboratory pipetting, inoculation, sampling, harvesting, extraction, processing and decontamination procedures. A typical site employs less than 10 workers/shift and operates 24 hours/day throughout the year. NIOSH has conducted walk-through surveys of several fermentation facilities in the enzyme industry and monitored for microbial air contamination. These particular facilities were not using recombinant microorganisms, but the processes were considered typical of fermentation process technology. Area samples were taken in locations where the potential for worker exposure was considered to be potentially greatest, i.e., near the fermentor, the seed fermentor, sampling ports, and separation processes (either filter press or rotary drum filter). The workers with the highest potential average exposures at the three facilities visited were those involved in air sampling. Area samples near the sampling port revealed average airborne concentrations ranging from 350 to 648 cfu/m3. Typically, the Chemical Engineering Branch would not use area monitoring data to estimate occupational exposure levels since the correlation between area concentrations and worker exposure is highly uncertain. Personal sampling data are not available at the present time. Thus, area sampling data have been the only means of assessing exposures for previous PMN biotechnology submissions. Assuming that 20 samples per day are drawn and that each sample takes up to 5 minutes to collect, the duration of exposure for a single worker will be about 1.5 hours/day. Assuming that the concentration of microorganisms in the worker's breathing zone is equivalent to the levels found in the area sampling, the worst-case daily inhalation exposure is estimated to range up to 650 to 1200 cfu/day. The uncertainty associated with this estimated exposure value is not known (Reilly, 1991).
Controversy exists over the ability to isolate A. oryzae from the natural environment. Some researchers believe that A. oryzae is widely distributed in nature while other maintain that all strains of A. oryzae are variants of A. flavus which have been modified through years of selection in an artificial environment. Specific data which indicates the survivability of industrial "domesticated" strains of A. oryzae in the environmentare not available. The process of domestication may have resulted in lessened survivability in the environment. However, its ability to produce spores suggests that it may survive in the environment (Versar, 1991).
Estimates of the number of A. oryzae organisms released during production are tabulated in Table 1 (Reilly, 1991). The uncontrolled/untreated scenario assumes no control features for the fermentor offgases, and no inactivation of the fermentation broth for the liquid and solid waste releases. The containment criteria required for the full exemption scenario assume the use of features or equipment that minimize the number of viable cells in the fermentor off-gases. They also assume inactivation procedures resulting in a validated 6log reduction of the number of viable microorganisms in the liquid and solid wastes relative to the maximum cell density of the fermentation broth.

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